ITC-based kinetics assay for NIS synthetases.

NIS Synthetases are a widely distributed, novel superfamily of enzymes critical to stealth siderophore production-small molecules increasingly associated with virulence. Study of these enzymes for inhibition or utilization in biosynthesis of new antibiotics has been hindered by multiple kinetics assays utilizing different limiting reporters or relying on product dissociation as a precursor to signal. We present a label free, continuous readout assay optimized for NIS Synthetase systems utilizing an isothermal titration calorimetry instrument.

Chemoenzymatic Synthesis of Select Intermediates and Natural Products of the Desferrioxamine E Siderophore Pathway.

The NIS synthetase family of enzymes responsible for the biosynthesis of siderophores is increasingly associated with bacterial virulence. Proteins in this class represent outstanding potential drug targets, assuming that basic biochemical and structural characterizations can be completed. Towards this goal, we have mated an improved synthesis of the non-commercial amino acid -hydroxy--succinylcadaverine (HSC, ) with an isothermal titration calorimetry (ITC) assay that profiles the iterative stages of HSC trimerization and macrocyclization by NIS synthetase DesD from .